Molecular cloning of anti-Müllerian hormone gene， polyclonal antibody preparation and response to exogenous estrogen in spotted steed （Hemibarbus maculatus Bleeker）

【Objective】In the present study, the anti-Müllerian hormone gene(Amh) in Hemibarbus maculatus was cloned. The sequence characteristics and subcellular localization were analyzed. Meanwhile, the expression changes under different concentrations of estrogen treatment were detected. The results will provide further theorial basis for the study of Amh gene function and its molecular mechanism in ovarian development.【Method】 The cDNA sequence of Amh gene was cloned and bioinformatically analyzed based on the transcriptome sequencing of spotted steed. The recombinant expression plasmid pET32a-Amh was construc-ted, transformed into Escherichia coli BL21, and polyclonal antibodies were prepared by immunizing mice with the purified recombinant protein. The subcellular localization of the protein was determined by immunofluorescence technique. Meanwhile, quantitative real-time PCR was used to detect the expression of Amh gene after estradiol treatment.【Result】The full length cDNA sequence of spotted steed Amh gene was 1849 bp, with an open reading frame(ORF) of 1671 bp that encoding 556 amino acid residues. Phylogenetic analysis showed that the spotted steed Amh gene clustered with Cyprinidae fish such as zebrafish and common carp, indicating that they were closely related. The purified AMH recombinant protein was immunized in mice to prepare AMH polyclonal antibody, and its antibody potency was above 2.4×10⁶ by enzyme-linked immunoassay(ELISA), and Western blotting showed that the antibody was specific.AMH protein was expressed in granulosa cells of spotted steed ovary. After treated with estradiol for 60 d, it was found that low concentration of estradiol(100 μg/g) promoted the growth of spotted steed oocytes and the number of cortical follicles. After treated with estradiol, the expression of Amh gene was reduced, indicating that estrogen inhibited Amh gene during ovary development.【Conclusion】Amh gene contains a structural domain typical of the TGF-β family, it is expressed in ovarian granulosa cells, and responds to estrogen regulation, and may play a regulatory role in spotted steed ovary development.